Adrenergic and cholinergic innervation of rat cerebral arteries

Summary A consecutive demonstration of both aminergic and cholinergic nerves of rat cerebral arteries was carried out on whole mount preparations. For demonstration of aminergic nerves the glyoxylic acid method was used, while for cholinergic nerves Karnovsky's technique was utilized consecutively. This procedure provided a highly sensitive and reproducible demonstration of the two systems on the same specimen. The results obtained were as follows: 1) Aminergic and cholinergic nerves were distributed densely in the proximal portions of the major cerebral arteries examined. They were dense in the arteries of the anterior circulation and sparse in the posterior circulation. Each nerve had two different patterns, i.e. circular and longitudinal. Both circular and longitudinal patterns were observed in the proximal portions of the arteries, while in the distal portions, longitudinal fibres were predominantly present. 2) Superior cervical ganglionectomy produced no change in cholinergic nerve distribution, while it caused decreased density of aminergic nerves on the ipsilateral side on the arteries of the anterior circulation. The contralateral anterior cerebral arteries were partially affected. Bilateral sympathectomies abolished aminergic nerves in all arteries except the vertebral artery.     

Histochemical studies on the regeneration of aminergic nerves in rat cerebral artery after superior cervical ganglionectomy

The course of regeneration of aminergic nerves in rat cerebral arteries was studied by means of histochemical methods, after uni- or bilateral cervical sympathectomy. Degeneration of aminergic nerves started on day 1 and was complete between days 3 and 7 after surgery. Between weeks 4 and 6, regenerating nerves started to appear from the proximal internal carotid artery. Regenerated aminergic nerve fibres were generally unbeaded and intensity of fluorescence was weak. The circular nerves appeared earlier than the longitudinal ones. The number of regenerating nerves reached the maximum, between months 9 and 12, at about half the normal level. AChE activity of the cerebral arteries showed no significant changes at any stage.     

Zinc-iodide-osmium procedures as markers of subcellular structures. I. Standardization of staining of transmitter containing vesicles

In the present investigation certain stain properties of the zinc iodide-osmium tetroxide mixture were investigated. It was observed that the type of reaction of certain cell structures with a ZIO mixture largely depended on several factors, namely, the pH of the mixture, aldehyde prefixation and type (s) of buffer (s) used. The standardization of these parameters led to the development of four procedures, each one of them with distinct stain properties. A nomenclature to designate these methods is proposed. The following procedures were applied to material processed for electron microscopy: 1. C.4.4-ZIO-4 degree -18 h: the ZIO mixture was prepared in citric acid-disodium phosphate buffer pH 4.4 and the tissue was incubated at 4 degree C during 18 H; 2. K-P.7.4-C.4.4-ZIO-4 degree -18 h: the tissue was prefixed in Karnovsky fixative prepared in phosphate buffer pH 7.4 and then incubated in C.4.4-ZIO at 4 degree C during 18 h; 3. V.7.4-ZIO-4 degree -18 H: the ZIO was prepared in veronal buffer pH 7.4 and incubation of the tissue was at 4 degree during 18 H; 4.K-P.7.4-V.7.4-ZIO-4 degree -18 h: the tissue was prefixed in Karnovsky fixative prepared in phosphate buffer pH 7.4 and then incubated in V.7.4-ZIO at 4 degree C during 18 h. The chromaffin cells and the cholinergic endings of the rat adrenal medulla and the vas deferens nerves were studied. C.4.4-ZIO-4 degree -18 h: This procedure stained adrenaline and noradrenaline storing granules. Synaptic vesicles at cholinergic endings were not stained. K-P.7.4.4-ZIO-4 degree -18 h: One type of chromaffin granule (probably storing noradrenaline) and both, the small and the granulated synaptic vesicles of cholinergic endings were deeply stained with this method. The aminergic fibres of the vas deferens reacted synaptic vesicles at cholinergic endings were not stained. K-P.7.4.4-ZIO-4 degree -18 h: One type of chromaffin granule (probably storing noradrenaline) and both, the small and the granulated synaptic vesicles of cholinergic endings were deeply stained with this method. The aminergic fibres of the vas deferens reacted synaptic vesicles at cholinergic endings were not stained. K-P.7.4.4-ZIO-4 degree -18 h: One type of chromaffin granule (probably storing noradrenaline) and both, the small and the granulated synaptic vesicles of cholinergic endings were deeply stained with this method. The aminergic fibres of the vas deferens reacted negatively. V.7.4-ZIO-4 degree -18 H: Both types of chromaffin granules and only the small synaptic vesicles of cholinergic endings were revealed with this procedure. In addition, some compartments of the Golgi complex were also stained. K-P.7.4-V.7.4-ZIO-4 degree -18 h: This method did not stain adrenaline and noradrenaline storing granules. Cholinergic synaptic vesicles appeared stained. However, the most striking stain property of this procedure was the staining of many cell organelles. The probable mechanisms by which different factors affect the ZIO reaction are discussed.     

Submucosal plexus of terminal ileum: a study of the cholinergic and noradrenergic nerves in rats with streptozotocin-induced diabetes.

In order to study the type and degree of the alterations in the innervation of the intestine in experimental diabetes, a histochemical study on the cholinergic and noradrenergic nerves of the submucosal plexus of terminal ileum from rats with streptozotocin-induced diabetes was performed. The results obtained suggest that the diabetic animals keep the cholinergic activity undamaged 20 weeks after the induction of the illness, while the number of the catecholaminergic nerves appears to be markedly reduced.     

Evidence for a cerebral cholinergic system and suggested pharmacological patterns of neural organization in the prostomium of the polychaete Nereis virens (SARS)

The histological visualization of choline acetyltransferase (CAT) and acetylcholinesterase (AChE) on frozen sections of prostomia of Nereis virens indicate a concentration of cholinergic activity in the anterior brain. Components are probably sensory epithelial cells with cholinergic axons entering the brain in cephalic nerves and efferent cholinergic axons to prostomial muscle leaving the brain in the same nerves. There are also subepidermal cholinergic cells that may be second order motor neurons serving epidermal mucous cells. The smaller, second lobe of the corpora pedunculata and its associated vertical fibre tract are CAT⁴ and appear continuous, on each side of the cerebral ganglion, with a dorsal and a ventral longitudinal bundle of AChE⁺ fibers. This system tapers to nothing at the level of the posterior eyes. There is a small AChE⁺ component to each optic nerve and AChE is present in the nuchal epithelium. These observations are discussed in relation to earlier studies on aminergic and neurosecretory activity in the same ganglion.     

Reinnervation of regenerating smooth muscle cells in minced vas deferens of the guinea-pig

Summary In adult guinea-pigs, a portion of the wall of the vas deferens was removed, minced and replaced. This caused muscle cells to dedifferentiate, divide and redifferentiate. Reinnervation of redifferentiating cells was followed using electron microscopy and histochemistry. Adrenergic nerves were first observed to re-enter the regenerating area 5 days after operation, and close contacts (within 20 nm) with muscle cells were first seen at 10 days. The total number of adrenergic nerves per 100 muscle cells reached control values by 5 weeks, and by 15 weeks was higher than control levels. Cholinergic nerves first appeared in the regenerating area about 3–4 weeks after the operation. The total number of cholinergic nerves present had not reached control values even at 15 weeks, and no nerve muscle contacts within 20 nm were observed. The ratio of adrenergic to cholinergic nerves in the regenerating area was higher at 15 weeks than in control tissue.This work was supported by grants from the Wellcome Trust and the Medial Research Council     

Intrinsic innervation of the ewe cervix and its variations during pregnancy

The course of the cholinergic and adrenergic nerve fibers in the cervix of the ewe was investigated in nonpregnant and pregnant animals using an acetylcholinesterase method and fluorescence histochemistry. Both technics in nonpregnant animals revealed a rich network of acetylcholinesterase and norepinephrine positive nerves around the blood vessels while the muscular innervation was moderately positive. Acetylcholinesterase fibers were also concentrated beneath the surface epithelium forming a plexus-like arrangement where isolated ganglion cells could be seen. At mid pregnancy cholinergic and adrenergic fibers decreased in density. The intensity of fluorescence was weaker and nerve fiber morphology was modified. We endeavoured to relate our findings to the problem of the neural control of contractions and the opening of the cervix of the ewe which is poorly supplied in nerve fibers, particularly at mid pregnancy.     

Intrinsic innervation of the ewe cervix and its variations during pregnancy

Summary The course of the cholinergic and adrenergic nerve fibers in the cervix of the ewe was investigated in nonpregnant and pregnant animals using an acetylcholinesterase method and fluorescence histochemistry. Both technics in nonpregnant animals revealed a rich network of acetylcholinesterase and norepinephrin positive nerves around the blood vessels while the muscular innervation was moderately positive. Acetylcholinesterase fibers were also concentrated beneath the surface epithelium forming a plexuslike arrangement where isolated ganglion cells could be seen. At mid pregnancy cholinergic and adrenergic fibers decreased in density. The intensity of fluorescence was weaker and nerve fiber morphology was modified. We endeavoured to relate our findings to the problem of the neural control of contractions and the opening of the cervix of the ewe which is poorly supplied in nerve fibers, particularly at mid pregnancy.     

The sphincter of the efferent filament artery in teleost gills: II. Sympathetic innervation

In addition to the cholinergic innervation described in the sphincter of the efferent filament arteries (Bailly and Dunel-Erb, ′86), an aminergic component has been demonstrated by specific techniques. The Falck fluorescence technique reveals a network of nerve fibers displaying a green fluorescence characteristic of catecholamines. At the ultrastructural level two types of fibers are present, one with clear vesicles and another with densecored vesicles. Axo-axonal synaptic relationships exist between the two types. Results of 5- and 6-OHDA (hydroxydopamine) treatments confirm the presence of an aminergic component. These observations support the notion of a dual innervation: cholinergic and adrenergic of, respectively, parasympathetic and sympathetic origin. The presence of presynaptic modulation is suggested. The aminergic component could inhibit or reduce the release of acetylcholine from cholinergic nerve endings. These results suggest that the sympathetic innervation modulates the vasoconstriction effect of the parasympathetic component.     

Localization of cholinergic innervation and neurturin receptors in adult mouse heart and expression of the neurturin gene

Neurturin (NRTN) is a neurotrophic factor required during development for normal cholinergic innervation of the heart, but whether NRTN continues to function in the adult heart is unknown. We have therefore evaluated NRTN expression in adult mouse heart and the association of NRTN receptors with intracardiac cholinergic neurons and nerve fibers. Mapping the regional distribution and density of cholinergic nerves in mouse heart was an integral part of this goal. Analysis of RNA from adult C57BL/6 mouse hearts demonstrated NRTN expression in atrial and ventricular tissue. Virtually all neurons in the cardiac parasympathetic ganglia exhibited the cholinergic phenotype, and over 90% of these cells contained both components of the NRTN receptor, Ret tyrosine kinase and GDNF family receptor α2 (GFRα2). Cholinergic nerve fibers, identified by labeling for the high affinity choline transporter, were abundant in the sinus and atrioventricular nodes, ventricular conducting system, interatrial septum, and much of the right atrium, but less abundant in the left atrium. The right ventricular myocardium contained a low density of cholinergic nerves, which were sparse in other regions of the working ventricular myocardium. Some cholinergic nerves were also associated with coronary vessels. GFRα2 was present in most cholinergic nerve fibers and in Schwann cells and their processes throughout the heart. Some cholinergic nerve fibers, such as those in the sinus node, also exhibited Ret immunoreactivity. These findings provide the first detailed mapping of cholinergic nerves in mouse heart and suggest that the neurotrophic influence of NRTN on cardiac cholinergic innervation continues in mature animals.This study was supported by the National Heart, Lung, and Blood Institute (grant HL-54633).     

Effects of infantile/prepubertal chronic estrogen treatment and chemical sympathectomy with guanethidine on developing cholinergic nerves of the rat uterus.

The innervation of the uterus is remarkable in that it exhibits physiological changes in response to altered levels in the circulating levels of sex hormones. Previous studies by our group showed that chronic administration of estrogen to rats during the infantile/prepubertal period provoked, at 28 days of age, an almost complete loss of norepinephrine-labeled sympathetic nerves, similar to that observed in late pregnancy. It is not known, however, whether early exposure to estrogen affects uterine cholinergic nerves. Similarly, it is not known to what extent development and estrogen-induced responses in the uterine cholinergic innervation are affected by the absence of sympathetic nerves. To address this question, in this study we analyzed the effects of infantile/prepubertal chronic estrogen treatment, chronic chemical sympathectomy with guanethidine, and combined sympathectomy and chronic estrogen treatment on developing cholinergic nerves of the rat uterus. Cholinergic nerves were visualized using a combination of acetylcholinesterase histochemistry and the immunohistochemical demonstration of the vesicular acetylcholine transporter (VAChT). After chronic estrogen treatment, a well-developed plexus of cholinergic nerves was observed in the uterus. Quantitative studies showed that chronic exposure to estrogen induced contrasting responses in uterine cholinergic nerves, increasing the density of large and medium-sized nerve bundles and reducing the intercept density of fine fibers providing myometrial and perivascular innervation. Estrogen-induced changes in the uterine cholinergic innervation did not appear to result from the absence/impairment of sympathetic nerves, because sympathectomy did not mimic the effects produced by estrogen. Estrogen-induced responses in parasympathetic nerves are discussed, considering the direct effects of estrogen on neurons and on changes in neuron-target interactions.     

Comparative study of the autonomic innervation of the mammalian ovary,with particular regard to the follicular system

Summary The autonomic innervation of the ovary was studied in 12 mammalian species utilizing the cholinesterase method in combination with pseudocholinesterase inhibition for the cholinergic component, and glyoxylic acid histochemistry together with fluorometric determination of noradrenaline for the adrenergic component. Ovaries from cow, sheep, cat, and guinea pig were very richly supplied with adrenergic nerves in the cortical stroma, particularly enclosing follicles in various stages of development. In the follicular wall the nerve terminals were located in the theca externa, where they ran parallel to the follicular surface. Numerous adrenergic terminals also surrounded ovarian blood vessels. The adrenergic innervation was of intermediary density in the human ovary and in the pig, dog, cat, and opossum. Ovaries from rabbit, mouse and hamster had a sparse adrenergic nerve supply. The amount of intraovarian adrenergic nerves agreed well with the tissue concentration of noradrenaline in the various species. The cholinergic innervation was generally less well developed, but had the same distribution as the adrenergic system around blood vessels and in the ovarian stroma, including follicular walls.     

Intrinsic innervation of the uterus in guinea pig and rat

The pattern of distribution of cholinergic and adrenergic nerves in the uterus of albino rats and guinea pigs was examined histochemically. In the albino rat, the uterus was found well-innervated by both adrenergic and cholinergic nerves with a clear regional variation. Dense innervation was demonstrated at the tubal and cervical ends of the uterus and in the cervix. Cholinergic nerves supplying the glands were more numerous than the adrenergic nerves which were relatively few. In the guinea-pigs, the uterus was richly innervated by adrenergic nerves with a clear regional variation. No cholinesterase-positive nerves or nerve cells were demonstrated.     

Pathogenesis of Thalidomide Teratogenesis in the Marmoset (Callithrix jacchus): Evidence Suggesting a Possible Trophic Influence of Cholinergic Nerves in Limb Morphogenesis

Thalidomide administered to the marmoset ( Callithrix jacchus ) will produce a pattern of deformities similar to those found in man. In the cervical and lumbar regions of the thalidomide malformed marmoset fetus, the spinal cord and dorsal root ganglia are smaller than those of the control. The reduction in size of the D.R.G.'s is due to a marked reduction in the number of neurons. Drugs which influence the function of cholinergic nerves in the chick and rabbit embryo interfere with development. Thalidomide produced peripheral neuropathy in adults by interference with the function of the cholinergic nerves. Thus it appears that thalidomide malformations are due in part, to an interference with numbers of, and probably the functions of, the cholinergic nerves in the embryo. It would appear that cholinergic nerves have a possible morphogenetic and trophic influence in embryogenesis.     

Immunohistochemical demonstration of VIP, [Met5]- and [Leu5]-enkephalin immunoreactive nerve fibres in the human prostate and seminal vesicles

Summary The distribution of nerves containing immunoreactivity for the VIP and enkephalins has been demonstrated in the human prostate and seminal vesicles using the immunoperoxidase bridge. VIP-containing nerves were detected in both organs studied mainly in association with the epithelium, while nerves containing ELI seemed to be related to smooth muscle. Compared with the distribution of adrenergic and cholinergic nerves in the prostate marked differences in the density of the innervation were detected. The possible nature of these peptide-containing nerves is discussed.     

A histochemical study on the innervation of the cerebral blood vessels in bats

Summary The adrenergic and cholinergic nerves innervating the cerebral blood vessels of four species of Japanese chiropterids (Rhinolophus ferrumequinum, Murina leucogaster, Vespertilio superans and Miniopterus Schreibersi) have been investigated using specific histochemical techniques. In all these species of bats arteries of the internal carotid system are poorly developed, whereas those of the vertebro-basilar system are well developed. The adrenergic and cholinergic nerves innervating these cerebral arteries, however, all originate from the stem nerve bundles entering the cranial cavity along the internal carotid artery. Both nerve plexuses are among the densest of any vertebrate species so far investigated. Adrenergic nerve plexuses are usually composed of complicated meshworks of fine fibres, while cholinergic ones are composed of rather longitudinally arranging meshworks of both thick and thin fibres, exhibiting a very high acetylcholinesterase activity. Small parenchymal arteries and arterioles are also dually innervated by adrenergic and cholinergic nerve fibres of peripheral origin. Intracerebral capillaries, on the other hand, are in several places directly connected with both adrenergic and cholinergic fibres of parenchymal origin. Capillaries in the cerebral and cerebellar cortices, diencephalon and cochlear nucleus in V. superans exhibit a heavy non-nervous acetylcholinesterase activity in their walls, but in R.ferrumequinum and M. schreibersi, the response is weak or negative, except for that in the cochlear nucleus.     

Characterization of postsynaptic β-adrenergic receptors and presynaptic muscarinic cholinergic receptors in the rat spleen

The β-adrenergic and muscarinic cholinergic receptors in the splenic homogenates of control and 6-hydroxydopamine (6-OHDA) treated rats were characterized. The specific binding of [³H]dihydroalprenolol (DHA) and [³H]quinuclidinyl benzilate (QNB) in the rat spleen were saturable and of high affinity and showed pharmacological specificity of splenic β-adrenergic and muscarinic cholinergic receptors. Following 6-OHDA treatment, the Bmax value for specific [³H](-)DHA binding to the rat spleen was significantly increased by 26 percent and 22 percent compared to control at 2 and 3 weeks without a change in the Kd. In contrast, there was a 38 percent decrease in the Bmax for [³H](-)QNB in the 6-OHDA treated rat spleen at 2 and 3 weeks respectively without a change in the Kd. The Bmax value at 5 weeks was significantly greater than that at 2 or 3 weeks. The splenic norepinephrine (NE) concentration was markedly reduced by the 6-OHDA treatment at 1 to 3 weeks, while there was a significant recovery in the splenic NE concentration at 5 weeks. Thus, our results strongly suggest that we are biochemically localizing muscarinic cholinergic receptors on the sympathetic nerves of the rat spleen and that the β-adrenergic receptors of the spleen are localized postsynaptically.     

Presence and distribution of cholinergic nerves in rat mediastinal brown adipose tissue.

Brown adipose tissue (BAT) is richly provided with sympathetic noradrenergic nerves but is believed to lack a parasympathetic nerve supply. Acetylcholine is the predominant transmitter of postganglionic parasympathetic nerves. The vesicular acetylcholine transporter (VAChT) resides in synaptic vesicles of cholinergic nerve terminals and is used as a marker for peripheral cholinergic nerves. We sought cholinergic nerves in rat BAT using VAChT immunohistochemistry (IHC) on cryosections of interscapular, cervical, mediastinal, and perirenal depots. Mediastinal BAT was the sole depot provided with putative parasympathetic perivascular and parenchymal cholinergic nerves. The absence of vasoactive intestinal peptide-positive nerves suggested their nature as pure cholinergic fibers. By confocal microscopy, both cholinergic and noradrenergic nerves were detected in mediastinal BAT. Cold exposure and fasting led to increased density of VAChT-positive fibers and of noradrenergic sympathetic nerves at morphometry. The unexpected double innervation of mediastinal BAT may explain the inhibitory influence on thermogenesis observed after systemic injection of muscarinic antagonists in rats, and raises questions about the physiological role of its cholinergic nerve supply.     

Cholinergic neurons of the pelvic autonomic ganglia and uterus of the female rat: distribution of axons and presence of muscarinic receptors

Acetylcholine (ACh) stimulates contraction of the uterus and dilates the uterine arterial supply. Uterine cholinergic nerves arise from the paracervical ganglia and were, in the past, characterized based on acetylcholinesterase (AChE) histochemistry. However, the histochemical reaction for acetylcholinesterase provides only indirect evidence of acetylcholine location and is a nonspecific marker for cholinergic nerves. The present study: (1) reevaluated cholinergic neurons of the paracervical ganglia, (2) examined the cholinergic innervation of the uterus by using retrograde axonal tracing and antibodies against molecules specific to cholinergic neurons, choline acetyltransferase and the vesicular acetylcholine transporter, and (3) examined muscarinic receptors in the paracervical ganglia using autoradiography and a radiolabeled agonist. Most ganglionic neurons were choline acetyltransferase- and vesicular acetylcholine transporter-immunoreactive and were apposed by choline acetyltransferase/vesicular acetylcholine transporter-immunoreactive terminals. Retrograde tracing showed that some cholinergic neurons projected axons to the uterus. These nerves formed moderately dense plexuses in the myometrium, cervical smooth muscle and microarterial system of the uterine horns and cervix. Finally, the paracervical ganglia contain muscarinic receptors. These results clearly reveal the cholinergic innervation of the uterus and cervix, a source of these nerves, and demonstrate the muscarinic receptor content of the paracervical ganglia. Cholinergic nerves could play significant roles in the control of uterine myometrium and vasculature.     

Decentralisation of neurones in the pelvic ganglion of the guinea-pig: Reinnervation by adrenergic nerves

An electron-microscopic study has been made of adrenergic and cholinergic nerve fibres and synapses in the pelvic ganglion of the guinea-pig at intervals of up to 60 days following section of the hypogastric and pelvic nerves. Transection of the hypogastric nerves led to degeneration of 80-90% of the cholinergic nerve profiles and synapses in the ganglion. The small number of adrenergic nerves and synapses did not change, but 30-60 days after section, this number increased 8-10 times. Transection of the pelvic nerves led to degeneration of about 15% of the cholinergic nerve terminals, but no change in adrenergic terminals. After transection of both hypogastric and pelvic nerves, only about 1% of cholinergic nerves survived, but after 30-60 days, the number of adrenergic nerves increased 8-10 times. It is concluded that following cholinergic nerve degeneration in the ganglion, adrenergic nerves, probably originating as collateral sprouts from postganglionic neurones and granule-containing cells, can replace them to some extent.